Methods for treating uterine disorders

ABSTRACT

A method for treating uterine disorders, including hyperplasic, hypertonic, cystic and/or neoplastic uterine gland tissue by local administration of a botulinum toxin to or to the vicinity of the afflicted uterine tissue.

CROSS REFERENCE

This application is a divisional of application Ser. No. 10/379,157filed Mar. 3, 2003, the entire contents of which application isincorporated herein by reference

BACKGROUND

The present invention relates to methods for treating uterine disorders.In particular, the present invention relates to methods for treatinguterine glandular disorders with a botulinum toxin.

An object of the present invention is to treat uterine tissues,including atypical uterine tissues, such as hyperplasic tissues,fibroids and uterine neoplasms (including tumors and cancers). A furtherobject of the present invention is to prevent the development of, or tocause the regression or remission of, atypical uterine tissues, fibroidsand neoplasms. An additional object of the present invention is to treatuterine disorders, both benign and cancerous, as well as for treatinghyperplasic and/or hypertonic uterine gland cells by localadministration of a Clostridial toxin to or to the vicinity of theafflicted uterine tissue.

Uterine Disorders

The uterus is a hollow muscular organ with significant glandular tissue.Upon release from the ovaries an egg travels through the Fallopian tubesto the uterus and if fertilized, the ovum embeds in the endometrium, aglandular lining of the uterus. The cervical canal extends from thevagina through the cervix (the lower portion of the uterus) to the bodyof the uterus. The fundus is the top of the uterus (the area between thefallopian tubes). The myometrium is the muscular wall of the uterus.

It is known that hyperplasic uterine tissues can, if not treated,develop into cancerous tissue. See e.g. Sivridis E. et al., Prognosticaspects on endometrial hyperplasia and neoplasia, Virchows Arch 2001August; 439(2):118-26. Additionally it is known that: differenthyperplasia, metaplasic or atypical breast tissues can develop intocancers (see e.g. Ellis I. O., et al, Tumors of the Breast, chapter 16(pages 865-930) of “Diagnostic Histopathology of Tumors”, volume 1,edited by Fletcher C. D. M., second edition, Churchill Livingstone(2000), discussed further infra, as well as Fabian C. J. et al Beyondtamoxifen new endpoints for breast cancer chemoprevention, new drugs forbreast cancer prevention,. Ann NY Acad Sci 2001 December; 952:44-59);hyperplasic intestinal tissues, such as polyps can transform intocarcinomas (see e.g. Der, R. et al Gastric Neoplasms, chapter 5 (pages105-144) of Chandraspma, P., “Gastrointestinal Pathology”, Appleton &Lange (1999), in particular pages 106-107; oral and oropharyngealepithelial hyperplasia indicates a precancerous lesion. Sunaga H., etal. Expression of granulocyte colony-stimulating factor receptor andplatelet-derived endothelial cell growth factor in oral andoropharyngeal precancerous lesions. Anticancer Res 2001 July-August;21(4B):2901-6, and; kidney and prostate cell hyperplasia has beendocumented as a factor leading to development of cancerous cells. VanPoppel, H., et al., Precancerous lesions in the kidney Scand J UrolNephrol Suppl 2000;(205):136-65.

Common cancers of the uterus include cervical and endometrial cancer.Endometrial cancer occurs most often in woman between the ages of 50 and70 and it more common in women who have not had children. The usualsymptom of endometrial cancer is vaginal bleeding after menopause.Diagnosis can be by biopsy or endometrial scraping.

Cervical cancer can take many years to develop. Before it does, earlychanges can occur in the cells of the cervix. The abnormal,non-cancerous cells (but which may become cancerous) are called cervicalintra-epithelial neoplasia (CIN) or dyskaryosis.

Smooth muscle tumors of the uterus can be submucosal, intramural, is andsubserosal leiomyomata (fibroids). Uterine leiomyomas (fibroids) of theuterus are one of the most common pathologic abnormalities of the femalegenital tract. Fibroids are typically mostly in the muscle of the uterus(intramural) and by virtue of their size or position can impinge uponthe endometrium and cause bleeding. Fibroids of the uterus are presentin about 25% of women and require treatment: (a) if due to position orsize they cause irregular uterine bleeding that cannot be controlledwith hormonal therapy or removal of a polyp-like fibroid (submucosal)from the inside of the uterus at time of hysteroscopy & D&C; (b) theyare so big (usually softball size or larger) that they give eitherpelvic pressure, bladder or rectal pressure or pelvic fullness symptoms;(c) they are in a position (usually near the ovaries or they have grownso rapidly that there is a question they might be malignant; (d) theycause recurrent pain due to the blood supply being compromised; (e) thefibroids cause distortion of the endometrial cavity and women haveproblems either during pregnancy or then they have frequent miscarriages

The location of fibroids is variable. Most commonly, they are intramuraland are noted by an irregular enlargement of the uterine corpus. Thetumors can enlarge from the surface of the uterus late or early in theircourse and become subserous. Alternatively, they can protrude into theendometrial cavity and distort it. The submucous fibroid is one that haspenetrated the endometrial cavity and has enlarged so as to stretch themucosa over the tumor to the point that the submucosa is absent andulceration of the overlapping endometrium may occur. Although not allsubmucous fibroids cause clinical bleeding or interfere with conceptionand normal pregnancy, they certainly are associated with significantsymptomatic disturbances of this type, exhibiting menorrhagia, anemia,pelvic cramping, infection, infertility, and abortion among the morecommonly seen problems.

Myomectomy removes the fibroid without removing the uterus.

Laparoscopic Myomectomy involves removing pedunculated subserosalfibroids through the navel and abdomen with the use of a laparoscope.Hysteroscopic Myomectomy involves the vaginal removal of submucosalfibroids through the use of a hysteroscope. Laparotomy (abdominalmyomectomy) involves an abdominal incision that allows for the removalof all fibroids no matter their location, size, or number. Laparoscopicmyomectomy with allows for the removal of slightly larger subserosalfibroids than what the laparoscope alone can handle and generallyincludes a relatively small incision of 3 inches or less in the abdomen.

Laparoscopic assisted vaginal myomectomy (LAVM) allows for thelaparoscopic removal of subserosal fibroids from the uterus with thetotal removal of fibroid material through a vaginal incision. Uterinefibroid embolization (UFE, also known as uterine artery embolizationUAE) is a minimally-invasive, non-surgical procedure performed by aninterventional radiologist (IR). This procedure involves placing acatheter into the artery and guiding it to the uterus. Small particlesare then injected into the artery. The particles block the blood supplyfeeding the fibroids.

Myolysis involves surgical instruments that are inserted through alaparoscopic incision in the abdomen and a high frequency electricalcurrent that is sent to the fibroid. The electrical current causes theblood vessels to vaso-constrict (become very small or close down) andthis basically cuts off the blood flow to the fibroids. The fibroidsremain in place and are not surgically removed. Without a blood supply,the fibroids eventually die and shrink.

There are three primary forms of hysterectomy. Subtotal, total andradical hysterectomy. Subtotal Hysterectomy involves only the removal ofthe uterus. The pelvic structural ligaments are not cut and the cervixis left in place. Fallopian tubes and ovaries may or may not be removed.This procedure is always done through the abdomen.

Total Hysterectomy involves removing both the body of the uterus and thecervix, which is the lower part of the uterus. It can sometimes be donethrough the vagina (vaginal hysterectomy); at other times, a surgicalincision in the abdomen is preferable. In a total hysterectomy andbilateral salpingo-oophorectomy, the ovaries and fallopian tubes areremoved, along with the uterus and cervix.

In radical hysterectomy the entire uterus and usually both tubes andovaries as well as the pelvic lymph nodes are removed through theabdomen.

In addition to the direct surgical risks, there may be longer-termphysical and psychological effects, potentially including depression andloss of sexual pleasure. If the ovaries are removed along with theuterus prior to menopause, there is an increased risk of osteoporosisand heart disease as well.

The surgical risks of hysterectomy and myomectomy include fever, bladderinfection and wound infection. A blood transfusion before surgery may benecessary because of anemia or during surgery for blood loss.Complications related to anesthesia may occur. Other complications caninclude blood clots, postoperative hemorrhage, bowel obstruction, injuryto the urinary tract and death (eleven women die for every 10,000hysterectomies performed).

Since clinically undetectable uterine cancer cells may be left followinglocal excision of the cancer, typically radiation therapy is given forlocal tumor control. Radiation therapy can also be used preoperativelyto shrink large uterine tumors and make them more easily resectable.Palliative radiation therapy is commonly used to relieve the pain ofbone metastasis and for the symptomatic management of metastases toother sites, such as the brain. Fatigue, skin reactions, changes insensation, color and texture of the skin, and uterine swelling arecommon during and immediately following a course of radiation therapy tothe uterus.

Chemotherapy, hormone therapy, or a combination of the two can be usedto palliate the effects of metastatic uterine disease.

Recommendations for adjuvant chemotherapy and/or adjuvant hormonetherapy are usually based on the number of positive axillary nodes,menopausal status, size of the primary tumor, and the estrogen receptorassay. The chemotherapeutic drugs most commonly used are alkylatingagents, antimetabolites, antitumor antibiotics (Herceptin) and vincaalkaloids. Hormone manipulation is achieved primarily through hormoneblockers and infrequently by surgical removal of sex hormone-producingglands (oophorectomy, adrenalectomy, or hypophysectomy). Tamoxifen, ananti-estrogen, is the most widely used hormonal agent. The second-linehormonal agents, such as Femara, and Arimidex, are now available forER/PR negative patients and/or patients who failed tamoxifen.Unfortunately, chemotherapy for uterine cancer can have numerousdeleterious side effects including fatigue, weight gain, nausea,vomiting, alopecia, disturbances in appetite and taste, neuropathies,diarrhea, bone marrow suppression, menopausal symptoms, hair loss andweight gain. Additionally, the first line drug of choice, tamoxifen, canincrease the risk of uterine cancer and blood clots.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.

The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating the isfoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of botulinum toxin (purified neurotoxincomplex) type A¹ is a LD₅₀ in mice. One unit (U) of botulinum toxin isdefined as the LD₅₀ upon intraperitoneal injection into female SwissWebster mice weighing 18-20 grams each. Seven immunologically distinctbotulinum neurotoxins have been characterized, these being respectivelybotulinum neurotoxin serotypes A, B, C₁, D, E, F and G each of which isdistinguished by neutralization with type-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that botulinum toxin type Ais 500 times more potent, as measured by the rate of paralysis producedin the rat, than is botulinum toxin type B. Additionally, botulinumtoxin type B has been determined to be non-toxic in primates at a doseof 480 U/kg which is about 12 times the primate LD₅₀ for botulinum toxintype A. Botulinum toxin apparently binds with high affinity tocholinergic motor neurons, is translocated into the neuron and blocksthe release of acetylcholine. ¹Available from Allergan, Inc., of Irvine,Calif. under the tradename BOTOX®.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A has been approved by the U.S. Food andDrug Administration for the treatment of blepharospasm, strabismus,hemifacial spasm and cervical dystonia. Non-type A botulinum toxinserotypes apparently have a lower potency and/or a is shorter durationof activity as compared to botulinum toxin type A. Clinical effects ofperipheral intramuscular botulinum toxin type A are usually seen withinone week of injection. The typical duration of symptomatic relief from asingle intramuscular injection of botulinum toxin type A averages aboutthree months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinum typesA and E both cleave the 25 kiloDalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 500 kD complex. Botulinum toxin type D is produced asboth 300 kD and 500 kD complexes. Finally, botulinum toxin types E and Fare produced as only approximately 300 kD complexes. The complexes (i.e.molecular weight greater than about 150 kD) are believed to contain anon-toxin hemaglutinin protein and a non-toxin and non-toxicnonhemaglutinin protein. These two non-toxin proteins (which is alongwith the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

It has been reported that botulinum toxin type A has been used inclinical settings as follows:

(1) about 75-250 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of diopter iscorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

-   -   (a) flexor digitorum profundus: 7.5 U to 30 U    -   (b) flexor digitorum sublimus: 7.5 U to 30 U    -   (c) flexor carpi ulnaris: 10 U to 40 U    -   (d) flexor carpi radialis: 15 U to 60 U    -   (e) biceps brachii: 50 U to 200 U. Each of the five indicated        muscles has been injected at the same treatment session, so that        the patient receives from 90 U to 360 U of upper limb flexor        muscle BOTOX® by intramuscular injection at each treatment        session.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Astudy of two commercially available botulinum type A preparations(BOTOX® and Dysport®) and preparations of botulinum toxins type B and F(both obtained from Wako Chemicals, Japan) has been carried out todetermine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀/ED₅₀. Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg forbotulinum toxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weaknessand duration were dose related for all serotypes. Water consumption wasgreater in mice injected with botulinum toxin type B than with BOTOX®,although botulinum toxin type B was less effective at weakening muscles.After four months of injections 2 of 4 (where treated with 1.5 ng/kg)and 4 of 4 (where treated with 6.5 ng/kg) rabbits developed antibodiesagainst botulinum toxin type B. In a separate study, 0 of 9 BOTOX®treated rabbits demonstrated antibodies against botulinum toxin type A.DAS results indicate relative peak potencies of botulinum toxin type Abeing equal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX® and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B.

It is known to use a botulinum toxin to treat: intrathecal pain (seee.g. U.S. Pat. No. 6,113,915); paragangliomas (see e.g. U.S. Pat. No.6,139,845); otic disorders (see e.g. U.S. Pat. No. 6,265,379);pancreatic disorders (see e.g. U.S. Pat. Nos. 6,143,306 and 6,261,572);migraine (see e.g. U.S. Pat. No. 5,714,468); smooth muscle disorders(see e.g. U.S. Pat. No. 5,437,291); prostate disorders, includingprostatic hyperplasia (see e.g. WO 99/03483 and Doggweiler R., et alBotulinum toxin type A causes diffuse and highly selective atrophy ofrat prostate, Neurourol Urodyn 1998; 17(4):363); autonomic nervedisorders, including hyperplasic sweat glands (see e.g. U.S. Pat. No.5,766,606); wound healing (see e.g. WO 00/24419); reduced hair loss (seee.g. WO 00/62746); skin lesions (see e.g. U.S. Pat. No. 5,670,484), and;neurogenic inflammatory disorders (see e.g. U.S. Pat. No. 6,063,768).U.S. Pat. No. 6,063,768 cursorily discloses at column 6 lines 39-42treatment of the inflammatory joint condition pigmented villonodularsynovitis and a particular type of joint cancer, synovial cell sarcoma.Column 6, line 53 of U.S. Pat. No. 6,063,768 also discloses, withoutfurther explanation, that “tumors” can be treated.

Additionally it has been disclosed that targeted botulinum toxins (i.e.with a non-native binding moiety) can be used to treat variousconditions (see e.g. U.S. Pat. No 5,989,545, as well as WO 96/33273; WO99/17806; WO 98/07864; WO 00/57897; WO 01/21213; WO 00/10598.

A botulinum toxin has been injected into the pectoral muscle to controlpectoral spasm. See e.g. Senior M., Botox and the management of pectoralspasm after subpectoral implant insertion, Plastic and Recon Surg, July2000, 224-225.

Both liquid stable formulations and pure botulinum toxin formulationshave been disclosed (see e.g. WO 00/15245 and WO 74703) as well astopical application of a botulinum toxin (see e.g. DE 198 52 981).

Acetylcholine

Typically or in general, only a single type of small moleculeneurotransmitter is released by each type of neuron in the mammaliannervous system. The neurotransmitter acetylcholine is secreted byneurons in many areas of the brain, but specifically by the largepyramidal cells of the motor cortex, by several different neurons in thebasal ganglia, by the motor neurons that innervate the skeletal muscles,by the preganglionic neurons of the autonomic nervous system (bothsympathetic and parasympathetic), by the postganglionic neurons of theparasympathetic nervous system, and by some of the postganglionicneurons of the sympathetic nervous system. Essentially, only thepostganglionic sympathetic nerve fibers to the sweat glands, thepiloerector muscles and a few blood vessels are cholinergic and most ofthe postganglionic neurons of the sympathetic nervous system secrete theneurotransmitter norepinephine. In most instances acetylcholine has anexcitatory effect. However, acetylcholine is known to have inhibitoryeffects at some of the peripheral parasympathetic nerve endings, such asinhibition of the heart by the vagus nerves.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic neurons of the parasympathetic nervoussystem, as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the synapses between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic. The nicotinicreceptors are also present in many membranes of skeletal muscle fibersat the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and insulin, respectively, from large dense-corevesicles. The PC12 cell line is a clone of rat pheochromocytoma cellsextensively used as a tissue culture model for studies ofsympathoadrenal development. Botulinum toxin inhibits the release ofboth types of compounds from both types of cells in vitro, permeabilized(as by electroporation) or by direct injection of the toxin into thedenervated cell. Botulinum toxin is also known to block release of theneurotransmitter glutamate from cortical synaptosomes cell cultures.

Wide Distribution of the Botulinum Toxin Substrate

It is known that a botulinum toxin can denervate muscle cells resultingin a flaccid paralysis due to a presynaptic inhibition of acetylcholinerelease from neurons at a neuromuscular junction. The proteolytic domainof a botulinum toxins acts upon a particular substrate in the cytosol oftarget cells, cleavage of the substrate preventing membrane docking andexocytosis of acetylcholine containing secretory vesicles. The absenceof acetylcholine in the synaptic cleft between innervating neuron andmuscle cell prevents stimulation of the muscle cells and paralysisthereby results.

The botulinum toxins are intracellular proteases that act specificallyon one or more of three different proteins which control the docking ofacetylcholine to containing secretory vesicles. These specificsubstrates for the botulinum toxins are synaptobrevin, syntaxin and/orSNAP-25. is See e.g. Duggan M. J., et al., A survey of botulinumneurotoxin substrate expression in cells, Mov Disorder 10(3);376:1995,and Blasi J., et al., Botulinum neurotoxin A selectively cleaves thesynaptic protein SNAP-25. Nature 365; 160-163:1993. For botulinum toxintypes B, D, F and G the particular intracellular substrate issynaptobrevin. SNAP-25, synaptobrevin and syntaxin are known as SNAREs(soluble N-ethylmaleimide sensitive factor attachment proteinreceptors).

Significantly, it is not only the nerves which innervate muscles whichcontain the substrate for the botulinum toxins: “The presence of SNAP-25in presynaptic regions of numerous neuronal subsets and in neural crestcell lines suggests that this protein subserves an important function inneuronal tissues.” Oyler G. A. et al., Distribution and expression ofSNAP-25 immunoreactivity in rat brain, rat PC-12 cells and humanSMS-KCNR neuroblastoma cells, Brain Res Dev Brain Res 1992 Feb. 21;65(2):133-146, 1992.

Additionally, “[T]he wide occurrence of the SNARE proteins in endocrinecells suggests that they may also serve as general diagnostic markersfor endocrine tumors . . . ”, Graff, L., et al. Expression of vesicularmonoamine transporters, synaptosomal-associated protein 25 andsyntaxin1: a signature of human small cell lung carcinoma, CancerResearch 61, 2138-2144, Mar. 1, 2001, at page 2138. For example, it isknown that SNAP-25 is widely distributed in neuroendocrine cells(including in chromaffin cells, PC12, GH3, and insulinomas).Furthermore, the botulinum toxin substrate synaptobrevin has been foundin fibroblasts and myeloid cells (e.g. mast cells). Duggan M., et al.,supra.

Indeed, SNAREs apparently influence or control the membrane fusion ofsecretory vesicles in most if not all secretory cells. Andersson J., etal, Differential sorting of SNAP-25a and SNAP-25b proteins inneuroblastoma cells, Eur J. Cell Bio 79, 781-789:November 2000.

Thus, the substrate for a botulinum toxin are not restricted to neuronalcells which release the neurotransmitter acetylcholine. The botulinumtoxin substrates are therefore “ubiquitously involved inmembrane-membrane fusion events” and the evidence points to “a universalmechanism for membrane fusion events” (i.e. for the docking of secretoryvesicles with the cell wall) (Duggan 1995, supra).

Thus, the intracellular substrate for botulinum toxin has a ubiquitousdistribution in both neuronal and non-neuronal secretory cells. This isclearly illustrated by discovery of the presence of SNAP-25 (a 25kiloDalton synaptosomal-associated protein and substrate for at leastbotulinum toxin type A) in at least:

-   (1) the pancreas (Sadoul K., et al., SNAP-25 is expressed in islets    of Langerhans and is involved in insulin release, J. Cell Biology    128; 1019-1029:1995;-   (2) the hypophysis (Dayanithi G., et al. Release of vasopressin from    isolated permeabilized neurosecretory nerve terminals is blocked by    the light chain of botulinum A toxin, Neuroscience 1990;    39(3):711-5);-   (3) the adrenal medulla (Lawrence G., et al. Distinct exocytotic    responses of intact and permeabilised chromaffin cells after    cleavage of the 25-kDa synaptosomal associated protein (SNAP-25) or    synaptobrevin by botulinum toxin A or B, Eur J. Biochem 236;    877-886:1996);-   (4) gastric cells (Hohne-Zell B., et al., Functional importance of    synaptobrevin and SNAP-25 during exocytosis of histamine by rat    gastric enterochromaffin-like cells, Endocrinology 138;    5518-5526:1997;-   (5) lung tumors (Graff, L., et al. Expression of vesicular monoamine    transporters, synaptosomal-associated protein 25 and syntaxin1: a    signature of human small cell lung carcinoma, Cancer Research 61,    2138-2144, Mar. 1, 2001 (small cell lung carcinomas (SCLCs) contain    SNAP-25);-   (6) intestinal tumors, Maksymowych A., et al., Binding and    transcytosis of botulinum neurotoxin by polarized human colon    carcinoma cells, J of Bio. Chem, 273 (34); 21950-21957: 1998    (botulinum toxin is internalized by human colon cancer cells);-   (7) pancreatic tumors, Huang, X., et al., Truncated SNAP-25 (1-197),    like botulinum neurotoxin A, can inhibit insulin secretion from    HIT-T15 insulinoma cells, Mol. Endo. 12(7); 1060-1070:1998(“ . . .    functional SNAP-25 proteins are required for insulin secretion . . .    ”, ibid. at page 1060). See also Boyd R., et al., The effect of    botulinum neurotoxins on the release of insulin from the insulinoma    cell lines HIT-15 and RINm5F, J. Bio Chem. 270(31);    18216-18218:1995, and; Cukan M., et al., Expression of SNAP-23 and    SNAP-25 in the pancreatic acinar tumor cell line AR42J, Molec Biol    Cell 20(suppl); 398a, no. 2305:1999 (“SNAP-25 is a SNARE protein    that mediates exocytotic events in neuronal and endocrine    systems.”);-   (8) pituitary tumors as well as in normal pituitary cells, Majo G.,    et al., Immunocytochemical analysis of the synaptic proteins SNAP-25    and Rab3A in human pituitary adenomas. Overexpression of SNAP-25 in    the mammososmatotroph lineages, J. Pathol 1997 December;    183(4):440-446;-   (9) neuroblastomas, Goodall, A., et al., Occurrence of two types of    secretory vesicles in the human neuroblastoma SH-SY5Y, J. of    Neurochem 68; 1542-1552:1997. See also Oyler, G. A, Distribution and    expression of SNAP-25 immunoreactivity in rat brain, rat PC-12 cells    and human SMS-KCNR neuroblastoma cells, Dev. Brain Res. 65 (1992);    133-146. Note that Goodall (1992) discusses only in vitro    identification of certain vesicle docking proteins in a single    neuroblastoma cell line;-   (10) kidney cells (Shukla A., et al., SNAP-25 associated    Hrs-2protein colocalizes with AQP2 in rat kidney collecting duct    principal cells, Am J Physiol Renal Physiol 2001 September;    281(3):F546-56 (SNAP-25 is involved in is kidney cell “regulated    exocytosis”), and;-   (11) normal lung cells (Zimmerman U. J., et al., Proteolysis of    synaptobrevin, syntaxin, and SNAP-25 in alveolar epithelial type II    cells, IUBMB Life 1999 October; 48(4): 453-8), and; (12) all ovarian    cells (Grosse J., et al., Synaptosome associated protein of 25    kilodaltons in oocytes and steroid producing cells of rat and human    ovary: molecular analysis and regulation by gonadotropins, Biol    Reprod 2000 August; 63(2): 643-50 (SNAP-25 found “in all oocytes and    in steroidogenic cells, including granulosa cells (GC) of large    antral follicles and luteal cells”

Cholinerqic Mammary Gland Tissues

Diverse hyperplasic and neoplastic mammary gland cells are influenced bycholinergic mechanisms. Thus, it has been discovered that there is a“cholinergic mechanism in the alveolar cells activity”. Balakina G. B.,et al., Localization of choline acetyltransferase in the alveolarportion of the mammary gland of the white mouse, Arkh Anat GistolEmbriol 1986 April; 90(4):73-7. Additionally, there is cholinergicinfluence upon both mammary dysplasia (fibrocysts) and mammary carcinomatissues (Dorosevich A. E., et al., Autonomic nerve endings and theircell microenvironment as one of the integral parts of the stromalcomponent in breast dysplasia and cancer, Arkh Patol 1994November-December; 56(6):49-53), as well as “a direct cholinergicstimulation of smooth muscle cells” in mammary arteries (Pesic S., etal., Acetylcholine-induced contractions in the porcine internal mammaryartery; possible role of muscarinic receptors, Zentralbl Veterinarmed A1999 October; 46(8): 509-15).

Significantly, an increase in acetylcholine due to inhibition ofcholinesterase has been implicated in increase mammary cellproliferation followed by the development of mammary carcinomas. CabelloG., et al, A rat mammary tumor model induced by the organo phosphorouspesticides parathion and malathion, possibly throughacetylcholinesterase inhibition, Environ Health Perspect 2001 May;109(5):471-9. Thus, a decrease in breast cancer cell proliferationappears to be mediated by a cholinergic mechanism. Panagiotou S.,“Opioid agonists modify breast cancer cell proliferation by blockingcells to the G2/M phase of the cycle: involvement of cytoskeletalelements, J Cell Biochem 1999 May 1; 73(2):204-11.

Adrenal Medulla

The adrenal or suprarenal glands are small, triangular-shaped structureslocated on top of the kidneys. Each adrenal gland comprises an adrenalcortex or outer portion and an adrenal medulla or inner portion. Thecortex surrounds and encloses the medulla.

The adrenal cortex secretes the hormones cortisol and aldosterone.Cortisol is produced during times of stress, regulates sugar usage, andis essential for maintenance of normal blood pressure. Aldosterone isone of the main regulators of salt, potassium and water balance. If bothadrenal glands are removed cortisol and aldosterone replacement therapyis mandatory.

The adrenal medulla secretes the catecholamines adrenalin (synonymouslyepinephrine) and noradrenalin (synonymously norepinephrine). Thesehormones are important for the normal regulation of a variety of bodilyfunctions, including stress reaction, when they cause an increase inblood pressure, the pumping ability of the heart, and the level of bloodsugar. Removal of the adrenal medulla results in little or no hormonaldeficiency because other glands in the body can compensate. Contrarily,excessive catecholamine production can be life threatening.

In the normal adult male about 85% of total catecholamine made by is theadrenal medulla is adrenaline, with the remaining 15% beingnoradrenalin. There is about 1.6 mg of catecholamine present per gram ofmedulla tissue. Most of the noradrenalin found in blood and urine comesnot from the adrenal medulla but from postganglionic sympathetic nerveendings. If the freshly sectioned adrenal gland is placed in fixativesthat contain potassium dichromate, the medulla turns brown and this isreferred to as the chromaffin reaction, so named to suggest the affinityof adrenal medulla tissue for chromium salts. Hence, cells of theadrenal medulla are often called chromaffin cells. Chromaffin cells alsoexists outside the adrenal medulla, but usually secrete onlynoradrenalin, not adrenaline.

The adrenal medulla can be viewed as a sympathetic ganglion innervatedby preganglionic cholinergic nerve fibers. These nerve fibers releaseacetylcholine which causes secretion of catecholamines (primarilyadrenaline) by a process of exocytosis from the chromaffin cells of theadrenal medulla. The normal adrenal medulla is innervated by thesplanchnic nerve, a preganglionic, cholinergic branch of the sympatheticnervous system. The activity of the adrenal medulla is almost entirelyunder such cholinergic nervous control.

Chromaffin Cell Tumors

Chromaffin cells (including the chromaffin cells of the adrenal medulla)and sympathetic ganglion cells have much in common as they are bothderived from a common embryonic ancestor, the sympathagonium of theneural crest, as shown diagrammatically below. Examples of the types ofneoplasms which can arise from each these cell types is shown inbrackets. Each of the cell types shown can potentially secretecatecholamines.

While most chromaffin cell neoplasms occur in the adrenal medulla,ectopic and multiple location chromaffin cell tumors are known,occurring most commonly in children.

1. Paragangliomas

A paraganglia (synonymously, chromaffin body) can be found in the heart,near the aorta, in the kidney, liver, gonads, and other places and iscomprised of chromaffin cells which apparently originate from neuralcrest cells and which have migrated to a close association withautonomic nervous system ganglion cells. A paraganglioma is a neoplasmcomprised of chromaffin cells derived from a paraganglia. A carotid bodyparaganglioma is referred to as a carotid paraganglioma, while anadrenal medulla paraganglioma is called a pheochromocytoma or achromaffinoma.

The carotid body is often observed as a round, reddish-brown to tanstructure found in the adventitia of the common carotid artery. It canbe located on the posteromedial wall of the vessel at its bifurcationand is attached by ayer's ligament through which the feeding vessels runprimarily from the external carotid. A normal carotid body measures 3-5mm in diameter. Afferent innervation appears to be provided through theglossopharyngeal nerve (the ninth cranial nerve). The glossopharyngealnerve supplies motor fibers to the stylopharyngeus, parasympatheticsecretomotor fibers to the parotid gland and sensory fibers to interalia the tympanic cavity, interior surface of the soft palate andtonsils). Histologically, the carotid body includes Type I (chief) cellswith copious cytoplasm and large round or oval nuclei. The cytoplasmcontains dense core granules that apparently store and releasecatecholamines. The normal carotid body is responsible for detectingchanges in the composition of arterial blood.

Carotid paragangliomas are rare tumors overall but are the most commonform of head and neck paraganglioma. The treatment of choice for mostcarotid body paragangliomas is surgical excision. However, because oftheir location in close approximation to important vessels and nerves,there is a very real risk of morbidity(mainly cranial nerve X-XIIdeficits and vascular injuries) and mortality which is estimated as3-9%. Tumor size is important because those greater than 5 cm indiameter have a markedly higher incidence of complications.Perioperative alpha and beta adrenergic blockers are given (if thecarotid paraganglioma is secreting catecholamines) or less preferablyangiographic embolization preoperatively. Radiotherapy, either alone orin conjunction with surgery, is a second consideration and an area ofsome controversy. Unfortunately, due to location and/or size,paragangliomas, including carotid paragangliomas can be inoperable.

2. Pheochromocytomas

Pheochromocytomas occur in the adrenal medulla and cause clinicalsymptoms related to excess catecholamine production, including suddenhigh blood pressure (hypertension), headache, tachycardia, excessivesweating while at rest, the development of symptoms after suddenlyrising from a bent-over position, and anxiety attacks. Abdominal imagingand 24 hour urine collection for catecholamines are usually sufficientfor diagnosis. Catecholamine blockade with phenoxybenzamine andmetyrosine generally ameliorates symptoms and is necessary to preventhypertensive crisis during surgery, the current therapy of choice.Standard treatment is laparoscopic adrenalectomy, although partialadrenalectomy is often used for familial forms of pheochromocytoma.Malignant (cancerous) pheochromocytomas are rare tumors.

Pheochromocytomas have been estimated to be present in approximately0.3% of patients undergoing evaluation for secondary causes ofhypertension. Pheochromocytomas can be fatal if not diagnosed or ifmanaged inappropriately. Autopsy series suggest that manypheochromocytomas are not clinically suspected and that the undiagnosedtumor is clearly associated with morbid consequences.

The progression of changes in the adrenal medulla can be from normaladrenal medulla to adrenal medullary hyperplasia (a generalized increasein the number of cells and size of the adrenal medulla without thespecific development of a tumor) to a tumor of the adrenal medulla(pheochromocytoma).

Treatment of a pheochromocytoma is surgical removal of one or bothadrenal glands. Whether it is necessary to remove both adrenal glandswill depend upon the extent of the disease. Patients who have had bothadrenal glands removed must take daily cortisol and aldosteronereplacement. Cortisol is replaced by either hydrocortisone, cortisone orprednisone and must be taken daily. Aldosterone is replaced by oraldaily fludrocortisone (Florineftm). Increased amounts of replacementhydrocortisone or prednisone are required by such patients duringperiods of stress, including fever, cold, influenza, surgical procedureor anesthesia.

3. Glomus Tumors

Glomus tumors (a type of paraganglioma) are generally benign neoplasms,also arising from neuroectodermal tissues, found in various parts of thebody. Glomus tumors are the most common benign tumors that arise withinthe temporal bone and fewer than five per cent of them is becomemalignant and metastasize. Glomus tumors arise from glomus bodiesdistributed along parasympathetic nerves in the skull base, thorax andneck. There are typically three glomus bodies in each ear. The glomusbodies are usually found accompanying Jacobsen's (CN IX) or Arnold's (CNX) nerve or in the adventitia of the jugular bulb. However, the physicallocation is usually the mucosa of the promontory(glomus tympanicums), orthe jugular bulb (glomus jugulare).

The incidence of glomus jugulare tumors is about 1:1,300,000 populationand the most striking bit of epidemiology is the predominant incidencein females with the female:male incidence ratio being at least 4:1.Catecholamine secreting (i.e. functional) tumors occur in about 1% to 3%of cases.

Glomus tumors have the potential to secrete catecholamines, similar tothe adrenal medulla which also arises from neural crest tissue and canalso secrete catecholamines. The neoplastic counterpart of a glomustumor in the adrenal gland is the pheochromocytoma, and glomus tumorshave been referred to as extra-adrenal pheochromocytoma. Catecholaminesecreting glomus tumors can cause arrhythmia, excessive perspiration,headache, nausea and pallor.

Glomus tumors can arise in different regions of the skull base. Whenconfined to the middle ear space, they are termed glomus tympanicum.When arising in the region of the jugular foramen, regardless of theirextent, they are termed glomus jugulare. When they arise high in theneck, extending towards the jugular foramen, they are termed glomusvagale. When they arise in the area of the carotid bifurcation, they arecalled carotid body tumors. Other known sites of glomus tumors includethe larynx, orbit, nose, and the aortic arch.

Glomus Jugulare tumors are the most common tumors of the middle ear.These tumors tend to be very vascular and are fed by branches of theexternal carotid artery. The symptoms of a glomus jugulare tumor includehearing loss with pulsatile ringing in the ear, dizziness, and sometimesear pain. The patient can have a hearing loss due possibly to blockageof the middle ear, but also there can be a loss of hearing due to nerveinjury from the tumor mass. Cranial nerve palsies of the nerves whichcontrol swallowing, gagging, shoulder shrugging and tongue movement canall be part of the presentation of glomus jugulare tumors. When thetympanic membrane is examined a red/blue pulsatile mass can often beseen. Symptoms are insidious in onset. Because of the location and thevascular nature of the tumors, a most common complaint is pulsatiletinnitus. It is believed that the tinnitus is secondary to mechanicalimpingement on the umbo is most cases. Other common symptoms are auralfullness, and (conductive) hearing loss.

Current therapy for a catecholamine secreting glomus tumor isirradiation and/or surgical ablation, preceded by administration ofalpha and beta blockers. Treatment for glomus jugulare tumors includesadministration of alpha and beta blockers. X-ray therapy can be used toimprove symptoms even if the mass persists. It is also possible toembolize the tumor with materials which block its blood supply, howeverthis procedure has associated problems with causing swelling of thetumor which can compress the brain stem and cerebellum as well asreleasing the catecholamines from the cells which die when they losetheir blood supply. Surgery can be carried out upon small tumorsappropriately located. The complications of surgery for a glomusjugulare tumor are persistent leakage of cerebrospinal fluid from theear and also palsy of one of the cranial nerves controlling facemovement, sensation or hearing.

Even though the surgery may be successful glomus jugulare tumors aresomewhat problematic because they have a high recurrence rate is and mayrequire multiple operations. Surgical ablation carries the risk ofmorbidity due mainly to iatrogenic cranial nerve deficits and CSF leaks.Lack of cranial nerve preservation is probably the most significantobjection to surgical intervention because of the associated morbidityof lower cranial nerve deficits. Radiotherapy also has seriouscomplications, including osteoradionecrosis of the temporal bone, brainnecrosis, pituitary-hypothalamic insufficiency, and secondarymalignancy. Other postoperative complications include CSF leaks,aspiration syndromes, meningitis, pneumonia and wound infections.

Thus, there are many deficiencies and drawbacks of the current therapiesfor benign uterine glandular afflictions and uterus cancers andhyperplasic tissues.

What is needed therefore is an effective, non-surgical ablation,non-radiotherapy therapeutic method for treating uterine glandularneoplasms and precancerous hyperplasic uterine tissues.

SUMMARY

The present invention meets this need and provides an effective,non-surgical ablation, non-radiotherapy therapeutic method for treatingvarious precancerous as well as cancerous uterine tissues. Thus, thepresent invention encompasses methods for treating atypical tissues,such as hyperplasic tissues, cysts and neoplasms (including tumors andcancers) and for preventing the development of, or for causing theregression or remission of, atypical uterine tissues, fibroids andneoplasms. In particular, the present invention encompasses methods fortreating uterine glandular disorders, uterine fibroids and neoplasms,both benign and cancerous, as well as for treating hyperplasic and/orhypertonic uterine cells by local administration of a Clostridial toxinto or to the vicinity of the afflicted the uterine tissue.

is An embodiment of the present invention is a method for treating auterine disorder by local administration of between about 10⁻³ U/kg andabout 2000 U/kg of a Clostridial neurotoxin to a uterine glandulartissue. The Clostridial neurotoxin can be a botulinum toxin. Preferably,the botulinum toxin is administered in an amount of between about 10⁻²U/kg and about 200 U/kg. More preferably, the botulinum toxin isadministered in an amount of between about 10⁻¹ U/kg and about 35 U/kg.The botulinum toxin is selected from the group consisting of botulinumtoxins types A, B, C, D, E, F and G and the preferred botulinum toxin isbotulinum toxin type A.

The local administration of the botulinum toxin can be carried out byimplantation of a botulinum toxin implant into or onto a uterine gland.The uterine gland disorder is selected from the group consisting ofprecancerous uterine tissue and uterine cancer. Thus, the uterinedisorder can be fibroids. The botulinum toxin can be locallyadministered by direct injection of the botulinum toxin into the uterineglandular tissue.

A more detailed embodiment of the present invention is a method fortreating a uterine gland disorder by local administration of betweenabout 10⁻³ U/kg and about 2000 U/kg of a botulinum toxin type A to auterine gland of a human patient, thereby a uterine gland disorder.

My invention also encompasses a method for treating a uterine glanddisorder by local administration of a botulinum toxin to a uterine glandor to the vicinity of a precancerous uterine tissue, thereby causing areduction in the size and/or activity of a hyperplastic, hypertonic orneoplastic uterine gland tissue. This method can reduce the diameter ofthe hyperplastic, hypertonic or neoplastic uterine gland tissue bybetween about 20% and about 100%, subsequent to the local administrationof the botulinum toxin.

Thus a method for treating a uterine gland disorder as disclosed hereincan comprise the step of local administration of a therapeutic amount ofa botulinum toxin to a hyperplastic, hypertonic or neoplastic uterinegland tissue, thereby causing a reduction in the diameter of thehyperplastic, hypertonic or neoplastic uterine gland tissue of betweenabout 20% and about 100%.

Additionally, the present invention encompasses a method for preventingdevelopment of a uterine gland neoplasm, the method comprising the stepof local administration of a botulinum toxin to a hyperplasic orhypertonic uterine gland tissue, thereby reducing a secretion from thehyperplasic or hypertonic uterine gland tissue and preventing thehyperplasic or hypertonic uterine gland tissue from developing into aneoplasm. In this method the botulinum toxin is administered in anamount of between about 10⁻³ U/kg and about 2,000 U/kg and the botulinumtoxin is selected from the group consisting of botulinum toxin types A,B, C, D, E, F and G. The botulinum toxin can be locally administered bydirect injection of the botulinum toxin into the hyperplasic orhypertonic uterine gland tissue.

To reiterate, a method for preventing development of a uterine glandneoplasm can comprise the step of local administration of a therapeuticamount of a botulinum toxin type A to the precancerous hyperplasic orhypertonic uterine gland tissue of a human patient, thereby preventingdevelopment of a uterine gland neoplasm.

Alternately, a method for preventing development of a uterine neoplasmcan comprise the step of local administration of between about 10⁻³ U/kgand about 2000 U/kg of a botulinum toxin to a hyperplasic tissue,wherein the botulinum toxin reduces a secretion from the hyperplasictissue by inhibiting a vesicle mediated exocytosis from is theprecancerous hyperplasic uterine tissue, thereby preventing developmentof the hyperplasic tissue into a neoplasm. The hyperplasic tissue cancomprise a substrate for the botulinum toxin selected from the group ofvesicle membrane docking proteins consisting of a 25 kiloDaltonsynaptosomal associated protein (SNAP-25), synaptobrevin and syntaxin.Furthermore, the botulinum toxin can be administered in an amount ofbetween about 1 U and about 40,000 U, such as between about 10⁻³ U/kgand about 35 U/kg, between about 10⁻² U/kg and about 25 U/kg, betweenabout 10⁻² U/kg and about 15 U/kg or between about 1 U/kg and about 10U/kg. and the local administration of the botulinum toxin is carried outby implantation of a botulinum toxin implant into or onto the body ofthe uterine tissue

A detailed embodiment of the present invention is a method forpreventing development of a uterine gland carcinoma (that is bypreventing the development of a benign [though hyperplasic, metaplasicor atypical] precancerous uterine tissue into a malignant neoplasm orcarcinoma), the method comprising the step of local administration ofbetween about 10⁻³ U/kg and about 2000 U/kg of a botulinum toxin type Ato a hyperplastic, metaplasic or atypical uterine tissue (such as anapocrine cell lined cyst) of a human patient, wherein the uterine tissuecomprises a substrate for the botulinum toxin selected from the group ofvesicle membrane docking proteins consisting of a 25 kiloDaltonsynaptosomal associated protein (SNAP-25), synaptobrevin and syntaxin,and wherein the botulinum toxin acts upon the substrate to reduce asecretion from the afflicted uterine tissue.

The present invention includes within its scope a method for treating aneoplasm by local administration of between about 10⁻³ U/kg and about2000 U/kg of a botulinum toxin to the neoplasm, thereby treating theneoplasm by either reducing the size of the neoplasm and/or by reducinga secretion from the uterine neoplasm.

A method according to the present invention can be carried out by directinjection of a botulinum toxin into the body of a neoplasm or byimplantation of a botulinum toxin implant into or onto the body of theuterine neoplasm. A method within the scope of the present invention canbe practiced to locally administer between about 10⁻³ U/kg and about2000 U/kg of a botulinum toxin to a neoplasm. U/kg means units of abotulinum toxin per kilogram of total patient weight. The botulinumtoxin can be one of the botulinum toxin types A, B, C₁, D, E, F and G,and is preferably a botulinum toxin type A because of the known clinicalefficacy of botulinum toxin type A for a number of indications andbecause of its ready availability.

Preferably, the botulinum toxin is administered in an amount of betweenabout 1 U and about 40,000 U (total units, not per kg of patientweight). At the higher dose ranges the amount of botulinum toxinadministered (i.e. 40,000 units) can be administered in the form of acontrolled release delivery system (i.e. an implant), whereby fractionalamounts of the botulinum toxin depot (i.e. about 10 units of a botulinumtoxin type A or about 500 units of a botulinum toxin type B) arereleased from the controlled release delivery system over a three tofour month period (continuous release delivery system) or is releasedfrom the controlled release delivery system in a multiphasic manner inapproximate three to four month repeating cycles (pulsatile releasedelivery system). Suitable controlled release delivery systems to use inthe present invention for either the continuous or pulsatile intra orperi-neoplasm release of therapeutic amounts of a botulinum toxin aredisclosed in U.S. Pat. Nos. 6,306,423 and 6,312,708.

In a more preferred embodiment of the present invention, the amount of abotulinum toxin type A locally administered to the body of or to a sitewithin the body of the uterine neoplasm according to the presentinvention can be an amount between about 10⁻³ U/kg and about 40 U/kg.Less than about 10⁻³ U/kg of a botulinum toxin type A is not expected toresult in a significant therapeutic efficacy, while more than about 40U/kg of a botulinum toxin type A can be expected to result in a toxic ornear toxic dose of the toxin. With regard to a botulinum toxin type B,the amount of a botulinum toxin type B locally administered to theneoplasm according to the present invention can be an amount betweenabout 10⁻³ U/kg and about 2000 U/kg. Less than about 10⁻³ U/kg of abotulinum toxin type B is not expected to result in a significanttherapeutic efficacy, while more than about 2000 U/kg of a botulinumtoxin type B can be expected to result in a toxic or near toxic dose ofthe type B toxin. It has been reported that about 2000 units/kg,intramuscular, of a commercially available botulinum toxin type Bpreparation approaches a primate lethal dose of type B botulinum toxin.Meyer K. E. et al, A Comparative Systemic Toxicity Study of Neurobloc inAdult and Juvenile Cynomolgus Monkeys, Mov. Disord 15(Suppl 2);54; 2000.With regard to the botulinum toxins types C, D, E, F and G, amounts forinjection into a neoplasm can be determined on a patient by patientbasis and are not expected to exceed the type B toxin dose range.

In a more preferred embodiment of the present invention, the amount of atype A botulinum toxin administered according to the disclosed methodsis between about 10⁻² U/kg and about 25 U/kg. Preferably, the amount ofa type B botulinum toxin administered by a continuous release systemduring a given period is between about 10⁻² U/kg and about 1000 U/kg,since it has been reported that less than about 1000 U/kg of type Bbotulinum toxin can be intramuscularly administered to a primate withoutsystemic effect. Ibid. More preferably, the type A botulinum toxin isadministered in an amount of between about 10⁻¹ U/kg and about 15 U/kg.Most preferably, the type A botulinum toxin is administered in an amountof between about 1 U/kg and about 10 U/kg. is In many instances, anintra-neoplastic administration of from about 1 units to less than about100 units of a botulinum toxin type A, can provide effective and longlasting therapeutic relief, as set forth herein. More preferably, fromabout 5 units to about 75 units of a botulinum toxin, such as abotulinum toxin type A, can be used and most preferably, from about 5units to about 50 units of a botulinum toxin type A, can be locallyadministered into a target neoplasm tissue with efficacious results. Ina particularly preferred embodiment of the present invention from about1 unit to about 50 units of a botulinum toxin, such as botulinum toxintype A, can be locally administered to a neoplasm target tissue withtherapeutically effective results, as described herein.

A detailed method within the scope of the present invention can becarried out by local administration of between about 10⁻³ U/kg and about2000 U/kg of a botulinum toxin type A to a neoplasm of a human patient,thereby reducing a secretion from the uterine neoplasm.

“Local administration” means direct injection of the neurotoxin into orto the local area of the target uterine tissue. Systemic routes ofadministration, such as oral and intravenous routes of administration,are excluded from the scope of the present invention.

The botulinum toxin can be a modified botulinum toxin, that is thebotulinum toxin can have at least one of its amino acids deleted,modified or replaced, as compared to a native botulinum toxin. Thus, thebotulinum toxin can be a recombinant produced botulinum toxin or aderivative or fragment thereof.

DRAWINGS

FIG. 1 is a diagramatic representation in partial cross section of thelocation of the uterus of a human female in relation to adjacent organs.

FIG. 2 is diagramatic cross sectional representation of the uterus ofFIG. 1.

FIG. 3 is diagramatic cross sectional representation of the uterus ofFIG. 2 showing location of different types of uterine fibroids.

DESCRIPTION

The present invention is based upon the discovery that hyperplasic,hypertonic, cystic and/or neoplastic uterine tissues can be treated witha Clostridial toxin to thereby reduce or eliminate the hyperplasia,hypertonia, cystic and/or neoplastic condition. The tissue treated canbe benign or malignant and hyperplasia includes a hypertonic condition.The present invention is therefore applicable to the treatment ofconditions which include uterine cancer, and fibroids, as well as tohyperplasic, metaplasic, atypia and dysplasic precancerous uterinetissues.

Additionally, excessively secreting uterine cells (hyperplasic orhypertonic) wherein the secretory activity is controlled or influencedby one or more of the botulinum toxin substrates can be treated by amethod within the scope of the present invention so as to prevent thedevelopment of the hyperplasic or hypertonic uterine secretory tissueinto a neoplasm. In the target tissue the proteolytic light chain of thebotulinum toxin is internalized.

Without wishing to be bound by theory, a physiological mechanism for theefficacy of the present invention can be proposed. Thus, it is knownthat uterine muscle tissue is influenced by cholinergic neurons. Seee.g. Morris, J., et al, Botulinum neurotoxin A attenuates release ofnorepinephrine but not NPY from vasoconstrictor neurons, Am J PhysiolHeart Circ Physiol 2002 December; 283(6). Additionally, it is known thatcholinergic innervation of uterine glandular tissues affects thesecretory activity of such cells. See e.g. Hammarstron M., et al., Doesnitric oxide act as a cellular messenger in muscarinic endometrialsecretion in the guinea-pig?, Acta Physiol Scand 2002 April;174(4):311-5. Thus, uterine secretory cells receive at least asympathetic cholinergic secremotor innervation. Hence it can bepostulated that local administration of a botulinum toxin to a uterineglandular tissue can act to reduce secretory activity by such glandularcells (either by inhibition of secretion promoting, cholinergicinnervation to the cells or by a direct effect upon of the toxin uponuterine glandular cells wherein toxin substrates SNAP-25 or VAMP promotemembrane docking or fusion of secretory vesicles), thereby reducing auterine glandular hyperplasia, which leads to a remission of fibroiddevelopment and furthermore inhibits progression or development of ahyperplasic (i.e. precancerous) uterine glandular cell into a uterinecancer, tumor or neoplasm.

In a preferred embodiment the present invention is a method for treatuterine disease, such as precancerous uterine tissues. Although thepresent invention is not limited to any particular mechanism, it can behypothesized that local administration of a Clostridial toxin (such as abotulinum toxin) to an afflicted tissue, such as a uterine fibroid,results in treatment of the i.e. fibroid (i.e. reduction of [or totalelimination of] size the fibroid, and/or of the uterine cellhyperplasia) due to either an inhibitory effect of the toxin uponstimulatory cholinergic fibers which innervate the uterine cells or adirect effect of the toxin upon the fibroid upon internalization of thetoxin (or at least of the toxin light chain) by fibroid cells.

Thus a preferred embodiment of the present invention is a method fortreating a precancerous uterine disorder, such as uterine fibroids, adenosis, papillomas, and fibroadenomas (hyperplasia lobules). Byprecancerous it is meant that the afflicted uterine tissue isnot-malignant (i.e. is not cancerous), although it can be hyperplasic,hypertrophic or metaplasic, and that the presence of the precanceroustissue increases the risk to the patient of development of a uterinecancer.

Thus, cholinergically innervated uterine target tissues can be treatedby local administration of a Clostridial toxin, such as a botulinumtoxin. By local administration it is meant that the neurotoxin isadministered directly into, or to the vicinity of the target tissue(i.e. a precancerous uterine tissue) or local tissue area to be treated.Local administration includes injection of the neurotoxin directly intothe afflicted tissue. Non-cancerous (benign), precancerous, cancerous(malignant) hyperplasic and/or hypertonic secretory tissues can betreated by a method within the scope of the present invention. Nodularor diffuse hyperplasia which precedes tumor development can be treatedby the present method.

I have discovered that a particular neurotoxin, botulinum toxin, can beused with dramatic ameliorative effect to treat a variety ofprecancerous uterine tissues, thereby significantly superseding currentsurgical, chemotherapy and radiotherapy therapeutic methods.Significantly, a single local administration of the botulinum toxin canbe used to successfully treat a uterine disease.

As shown by FIG. 1, the uterus is situated in proximity to the bladderand the intestine. FIG. 2 shows that the uterus comprises a fundus,body, cervix, cervical canal, endometrium and myometrium. Various typesof fibroids are possible, including intramural, pedunculated submucosal,pedunculated subserosal, submucosal, and subserosal fibroids and typicaluterine location are shown by FIG. 3.

The route of administration and amount of botulinum toxin administeredcan vary widely according to the particular uterine gland disorder beingtreated and various patient variables including size, weight, age,disease severity and responsiveness to therapy. Method for determiningthe appropriate route of administration and dosage are generallydetermined on a case by case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see forexample, Harrison's Principles of Internal Medicine (1997), edited byAnthony Fauci et al., 14^(th) edition, published by McGraw Hill).Treatment is carried out so as to substantially avoiding entry of thetoxin into the systemic circulation (i.e. by use of subcutaneous orintramuscular injection as opposed to intravenous administration).

The specific dosage appropriate for administration is readily determinedby one of ordinary skill in the art according to the factors discussedabove. The dosage can also depend upon the size of the tumor to betreated or denervated, and the commercial preparation of the toxin.Additionally, the estimates for appropriate dosages in humans can beextrapolated from determinations of the amounts of botulinum requiredfor effective denervation of other non-neoplastic tissues. Thus, theamount of botulinum A to be injected is proportional to the mass andlevel of activity of the uterine tissue to be treated. Generally,between about 0.01 and 2000 units per kg of patient weight of abotulinum toxin, such as botulinum toxin type A, can be administered toeffectively accomplish a toxin induced target tissue atrophy uponadministration of the neurotoxin at or to the vicinity of the uterinetarget tissue. Less than about 0.01 U/kg of a botulinum toxin does nothave a significant therapeutic effect while more than about 2000 U/kg or35 U/kg of a botulinum toxin B or A, respectively, approaches a toxicdose of the specified botulinum toxin. Careful placement of theinjection needle and a low volume of neurotoxin used preventssignificant amounts of botulinum toxin from appearing systemically. Amore preferred dose range is from about 0.01 U/kg to about 25 U/kg of abotulinum toxin, such as that formulated as BOTOX®. The actual amount ofU/kg of a botulinum toxin to be administered depends upon factors suchas the extent (mass) and level of activity of the i.e. hyperplasicuterine tissue to be treated and the administration route chosen.Botulinum toxin type A is a preferred botulinum toxin serotype for usein the methods of the present invention.

The main site of action of botulinum toxin is the neuromuscular junctionwhere the toxin binds rapidly and prevents the release of acetylcholine.Thus, while it is known that the botulinum toxins have a known bindingaffinity for cholinergic, pre-synaptic, peripheral motor neurons, I havediscovered that the botulinum toxins can also bind to and translocateinto a variety of precancerous uterine tissues, where the toxin thenacts, in the known manner, as an endoprotease upon its respectivesecretory vessel-membrane docking protein. Because of the lower affinityof the botulinum toxins for certain uterine tissues, the toxin canpreferably injected into secretory or glandular tissues to provide ahigh local concentration of the toxin. Thus, the present invention isapplicable to the treatment of precancerous uterine tissues which mayhave with little or no cholinergic innervation.

Preferably, a neurotoxin used to practice a method within the scope ofthe present invention is a botulinum toxin, such as one of the serotypeA, B, C, D, E, F or G botulinum toxins. Preferably, the botulinum toxinused is botulinum toxin type A, because of its high potency in humans,ready availability, and known use for the treatment of skeletal andsmooth muscle disorders when locally administered by intramuscularinjection.

A route for administration of a neurotoxin according to the presentdisclosed invention for treating a precancerous uterine tissue can beselected based upon criteria such as the solubility characteristics ofthe neurotoxin toxin chosen as well as the amount of the neurotoxin tobe administered. The amount of the neurotoxin administered can varywidely according to the particular disorder being treated, its severityand other various patient variables including size, weight, age, andresponsiveness to therapy. For example, the extent of the precancerousuterine tissue influenced is believed to be proportional to the volumeof neurotoxin injected, while the quantity of the denervation is, formost dose ranges, believed to be proportional to the concentration ofneurotoxin injected. Methods for determining the appropriate route ofadministration and dosage are generally determined on a case by casebasis by the attending physician. Such determinations are routine to oneof ordinary skill in the art (see for example, Harrison's Principles ofInternal Medicine (1997), edited by Anthony Fauci et al., 14th edition,published by McGraw Hill).

The present invention includes within its scope the use of anyneurotoxin which has a long duration therapeutic effect when locallyapplied to a precancerous uterine tissue of a patient. For example,neurotoxins made by any of the species of the toxin producingClostridium bacteria, such as Clostridium botulinum, Clostridiumbutyricum, and Clostridium beratti can be used or adapted for use in themethods of the present invention. Additionally, all of the botulinumserotypes A, B, C, D, E, F and G can be advantageously used in thepractice of the present invention, although type A is the most preferredserotype, as explained above. Practice of the present invention canprovide target tissue atrophy and remission for 27 months or longer inhumans.

It is known that catecholamine release from permeabilized adrenalmedulla cells can be inhibited by a botulinum toxin. Additionally, it isknown that release of insulin from permeabilized (as by electroporation)insulin secreting cells can be inhibited by a botulinum toxin. When invitro, the cell membranes of these non-nerve cells can be permeabilizedto assist introduction of a botulinum toxin into the cell's cytosol dueto the lack of cell surface receptors for a botulinum toxin. Thus,botulinum toxin type B apparently inhibits insulin secretion by cleavingsynaptobrevin present in the insulin secreting cell line HIT-15. Boyd R.S., et al The Effect of Botulinum Neurotoxin-B On Insulin Release From aBeta Cell, Mov Disord 10(3):376 (1995). It is the inventor's contentionthat a botulinum toxin can block the release of any vesicle mediatedexocytosis from any secretory (i.e. neuronal, glandular, secretory,chromaffin) cell type, as long as the light chain of the botulinum toxinis translocated into the intracellular medium. For example, theintracellular protein SNAP-25 is widely distributed in both neuronal andnon-neuronal secretory cells and botulinum toxin type A is anendopeptidase for which the specific substrate is SNAP-25. Thus, whilecholinergic neurons have a high affinity acceptor for the botulinum andtetanus toxins (and are therefore more sensitive than other neurons andother cells to the inhibition of vesicle mediated exocytosis ofsecretory compounds), as the toxin concentration is raised,non-cholinergic sympathetic neurons, chromaffin cells and other celltypes can take up a botulinum toxin and show reduced exocytosis.

Hence, by practice of the present disclosed invention, non-cholinergicnerve fibers as well as non or poorly innervated secretory uterineneoplasms can be treated by use of an appropriately higher concentrationof a botulinum toxin to bring about therapeutic atrophy of secretoryuterine neoplasms.

Furthermore, a method within the scope of the present invention canprovide improved patient function. “Improved patient function” can bedefined as an improvement measured by factors such as a reduced pain,reduced time spent in bed, increased ambulation, healthier attitude,more varied lifestyle and/or healing permitted by normal muscle tone.Improved patient function is synonymous with an improved quality of life(QOL). QOL can be assesses using, for example, the known SF-12 or SF-36health survey scoring procedures. SF-36 assesses a patient's physicaland mental health in the eight domains of physical functioning, rolelimitations due to physical problems, social functioning, bodily pain,general mental health, role limitations due to emotional problems,vitality, and general health perceptions. Scores obtained can becompared to published values available for various general and patientpopulations.

As set forth above, I have discovered that a surprisingly effective andlong lasting therapeutic effect can be achieved by local administrationof a neurotoxin to a precancerous uterine tissue of a human patient. Inits most preferred embodiment, the present invention is practiced bydirect injection into the target tissue or to the local area of thetarget tissue of botulinum toxin type A. It has been reported that atthe neuroglandular junction, the chemical denervation effect of abotulinum toxin, such as botulinum toxin type A, has a considerablylonger duration of action, i.e. 27 months vs. 3 months.

The present invention does include within its scope: (a) neurotoxincomplex as well as pure neurotoxin obtained or processed by bacterialculturing, toxin extraction, concentration, preservation, freeze dryingand/or reconstitution and; (b) modified or recombinant neurotoxin, thatis neurotoxin that has had one or more amino acids or amino acidsequences deliberately deleted, modified or replaced by knownchemical/biochemical amino acid modification procedures or by use ofknown host cell/recombinant vector recombinant technologies, as well asderivatives or fragments of neurotoxins so made, and includesneurotoxins with one or more attached targeting moieties for chromaffinand neoplasm cells types.

Botulinum toxins for use according to the present invention can bestored in lyophilized or vacuum dried form in containers under vacuumpressure. Prior to lyophilization the botulinum toxin can be combinedwith pharmaceutically acceptable excipients, stabilizers and/orcarriers, such as albumin. The lyophilized or vacuum dried material canbe reconstituted with saline or water.

In each of the following examples, the specific amount of a botulinumtoxin administered depends upon a variety of factors to be weighed andconsidered within the discretion of the attending physician and in eachof the examples insignificant amounts of botulinum toxin enter appearsystemically with no significant side effects. Units of botulinum toxininjected per kilogram (U/kg) below are per kg of total patient weight.For example, 3 U/kg for a 70 kg patient calls for an injection of 210units of the botulinum toxin.

EXAMPLES

The following examples provide those of ordinary skill in the art withspecific preferred methods within the scope of the present invention forcarrying out the present invention and are not intended to limit thescope of what the inventors regards as their invention.

In each of the following examples, the specific amount of a botulinumtoxin (such as BOTOX®) administered depends upon a variety of factors tobe weighed and considered within the discretion of the attendingphysician.

Example One Use of a Botulinum Toxin to Treat Fibroids

A 46 year old female presents with uterine fibroids. Ultrasound andimaging investigation reveals multiple fibroids. Histologicalexamination reveals the present of endometrial atypia (both hyperplasiaand metaplasia) and the patient is therefore determined to be at riskfor development of carcinoma. Local administration (injection) of from10 unit to 100 units of a botulinum toxin type A, such as BOTOX®′ intothe fibroid mass at several locations is carried out. Within 28 daysthereafter the fibroids have substantially regressed (fibroid diameterreduced by at least 80%) and remains so for the ensuing 2 to 24 months.Alternately, a botulinum toxin type B, C, D, E, F or G can beadministered, with the dosing amount adjusted to reflect the differingpotency as compared to the type A toxin. Thus, for example, sincebotulinum toxin type B is known to be about 50 times less potent thatbotulinum toxin type A, from 500 to 5000 unit of type B toxin is localadministered. Alternately, for extended therapeutic effect, a controlledrelease implant can be inserted subcutaneously and/or a suspension ofbotulinum containing microspheres can be injected, as set forth in U.S.Pat. Nos. 6,306,423 and 6,312,708.

Example 2 Treatment of Uterine Hypertonic or Hyperplasic Tissues with aBotulinum Toxin

A 64 year old woman is diagnosed with precancerous, hyperplasic uterinetissues. Local administration (injection) of from 10 unit to 100 unitsof a botulinum toxin type A, such as BOTOX®′ into the hyperplasictissues is carried out. Within 28 days thereafter the hyperplasia havesubstantially regressed and remains so for the ensuing 2 to 24 months.Alternately, a botulinum toxin type B, C, D, E, F or G can beadministered, with the dosing amount adjusted to reflect the differingpotency as compared to the type A toxin. Thus, for example, sincebotulinum toxin type B is known to be about 50 times less potent thatbotulinum toxin type A, from 500 to 5000 unit of type B toxin is localadministered. Alternately, for extended therapeutic effect, a controlledrelease implant can be inserted subcutaneously and/or a suspension ofbotulinum containing microspheres can be injected, as set forth in U.S.Pat. Nos. 6,306,423 and 6,312,708. The same local administration methodcan be carried out to treat a uterine metastatic lesion, as wells aspreoperative prior to surgical removal of a hyperplasic, or cancerousuterine tissue.

Methods according to the invention disclosed herein has many advantages,including the following:

(1) the invention renders unnecessary surgery for effective treatment ofdiverse uterine diseases, including hyperplasic, hypertonic andmetaplasic uterine tissues.

(2) systemic drug effects can be avoided by direct local application ofa neurotoxin according to the present invention

(3) the ameliorative effects of the present invention can persists fortwo years or longer from a single local administration of a neurotoxinas set forth herein.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes local otic administration methods wherein two or moreneurotoxins, such as two or more botulinum toxins, are administeredconcurrently or consecutively. For example, botulinum toxin type A canbe administered until a loss of clinical response or neutralizingantibodies develop, followed by administration of botulinum toxin typeE. Alternately, a combination of any two or more of the botulinumserotypes A-G an be locally administered to control the onset andduration of the desired therapeutic result. Furthermore, non-neurotoxincompounds can be administered prior to, concurrently with or subsequentto administration of the neurotoxin to proved adjunct effect such asenhanced or a more rapid onset of denervation before the neurotoxin,such as a botulinum toxin, begins to exert its therapeutic effect.

My invention also includes within its scope the use of a neurotoxin,such as a botulinum toxin, in the preparation of a medicament for thetreatment of a precancerous uterine tissue by local administration ofthe neurotoxin. All references, articles, publications and patents citedabove are incorporated herein by reference in their entireties.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

I claim:
 1. A method for treating a uterine glandular disorder, themethod comprising the step of local administration of between about 10⁻³U/kg and about 2000 U/kg of a Clostridial neurotoxin to a uterine gland,thereby treating a uterine gland disorder.
 2. The method of claim 1,wherein the Clostridial neurotoxin is a botulinum toxin.
 3. The methodof claim 2, wherein the botulinum toxin is administered in an amount ofbetween about 10⁻² U/kg and about 200 U/kg.
 4. The method of claim 2,wherein the botulinum toxin is administered in an amount of betweenabout 10⁻¹ U/kg and about 35 U/kg.
 5. The method of claim 2, wherein thebotulinum toxin is selected from the group consisting of botulinumtoxins types A, B, C, D, E, F and G.
 6. The method of claim 2, whereinthe botulinum toxin is botulinum toxin type A.
 7. The method of claim 2,wherein local administration of the botulinum toxin is carried out byimplantation of a botulinum toxin implant into or onto a uterine gland.8. The method of claim 1, wherein the uterine gland disorder is selectedfrom the group consisting of precancerous uterine tissue and uteruscancer.
 9. A method for treating a uterine gland disorder, the methodcomprising the step of local administration of between about 10⁻³ U/kgand about 2000 U/kg of a botulinum toxin type A to a uterus of a humanpatient, thereby treating a uterine gland disorder by reducing asecretion from the uterine gland and treating the uterine glanddisorder.
 10. A method for treating a uterine fibroid, the methodcomprising the step of local administration of between about 10⁻³ U/kgand about 2000 U/kg of a botulinum toxin to a uterine fibroid, therebytreating the uterine fibroid.
 11. A method for treating a uterine glanddisorder, the method comprising the step of local administration of abotulinum toxin to a uterine gland or to the vicinity of a precancerousuterus tissue, thereby causing a reduction in the size and/or activityof the hyperplastic, hypertonic or neoplastic uterine gland tissue. 12.A method for treating a uterine gland disorder, the method comprisingthe step of local administration of a therapeutic amount of a botulinumtoxin to a hyperplastic, hypertonic or neoplastic uterine gland tissue,thereby causing a reduction in the diameter of the hyperplastic,hypertonic or neoplastic uterine gland tissue of between about 20% andabout 100%.
 13. A method for preventing development of a uterine glandneoplasm, the method comprising the step of local administration of abotulinum toxin to a hyperplasic or hypertonic uterine gland tissue,thereby reducing a secretion from the hyperplasic or hypertonic uterinegland tissue and preventing the hyperplasic or hypertonic uterine glandtissue from developing into a neoplasm.
 14. A method for preventingdevelopment of a uterine neoplasm, the method comprising the step oflocal administration of a therapeutic amount of a botulinum toxin type Ato the precancerous hyperplasic or hypertonic uterine gland tissue of ahuman patient, thereby preventing development of a uterine glandneoplasm.
 15. The method of claim 14 wherein the hyperplasic uterinetissue comprises a substrate for the botulinum toxin selected from thegroup of vesicle membrane docking proteins consisting of a 25 kiloDaltonsynaptosomal associated protein (SNAP-25), synaptobrevin and syntaxin.16. A method for preventing development of a uterine carcinoma, themethod comprising the step of local administration of between about 10⁻³U/kg and about 2000 U/kg of a botulinum toxin type A to a hyperplasticuterus tissue of a human patient, wherein the hyperplastic uterus tissuecomprises a substrate for the botulinum toxin selected from the group ofvesicle membrane docking proteins consisting of a 25 kiloDaltonsynaptosomal associated protein (SNAP-25), synaptobrevin and syntaxin,and wherein the botulinum toxin acts upon the substrate to reduce asecretion from the hyperplasic uterus tissue.